(<40 and 40) and screening ppfev 1. , were done for patients with baseline ppfev 1

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1 Efficacy and safety of lumacaftor/ivacaftor combinion therapy in pients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis J Stuart Elborn, Bonnie W Ramsey, Michael P Boyle, Michael W Konstan, Xiaohong Huang, Gautham Marigowda, David Waltz, Claire E Wainwright, for the VX-809 TRAFFIC and TRANSPORT study groups Summary Background Lumacaftor/ivacaftor combinion therapy has shown clinical benefits in pients with cystic fibrosis homozygous for the Phe508del CFTR mution; however, pretrement lung function is a confounding factor th potentially affects the efficacy and safety of this therapy. We aimed to assess the efficacy and safety of lumacaftor/ ivacaftor therapy in these pients, defined by specific cegories of lung function. Methods Both trials (TRAFFIC and TRANSPORT) included in this pooled analysis were multinional, randomised, double-blind, placebo-controlled, parallel-group, phase 3 studies. Eligible pients from 187 participing centres in North America, Australia, and the European Union (both trials) were aged 12 years or older with a confirmed diagnosis of cystic fibrosis, homozygous for the Phe508del CFTR mution, and with a percent predicted FEV 1 ( ) of the time of screening. Pients were randomly assigned with an interactive web response system (1:1:1) to receive placebo, lumacaftor (600 mg once daily) plus ivacaftor (250 mg every 12 h), or lumacaftor (400 mg every 12 h) plus ivacaftor (250 mg every 12 h) for 24 weeks. Prespecified subgroup analyses of pooled efficacy and safety da by lung function, as measured by, were done for pients with (<40 and 40) and screening (<70 and 70). The primary endpoint was the absolute change from in week 24 analysed in all randomised pients who received least one dose of study drug. Both trials are registered with ClinicalTrials. gov (TRAFFIC: NCT ; TRANSPORT: NCT ). Findings Both trials were done between April, 2013, and April, Of the 1108 pients included in the efficacy analysis, 81 pients had a th decreased to lower than 40 between screening and and 1016 had a of 40 or higher. At screening, 730 had a of less than 70, and 342 had a of 70 or higher. Improvements in the absolute change from week 24 in were observed with both lumacaftor/ivacaftor doses in the subgroup with levels lower than 40 (least-squares difference vs placebo was 3 7 percentage points [95% CI ; p=0 024] in the lumacaftor [600 mg/day] ivacaftor group and 3 3 percentage points [ ; p=0 036] in the lumacaftor [400 mg/12 h] ivacaftor group). Improvements in compared with placebo were also reported in the subgroup with levels of 40 or higher (3 3 percentage points [ ; ] in the lumacaftor [600 mg per day] ivacaftor group and 2 8 percentage points [ ; ] in the lumacaftor [400 mg/12 h] ivacaftor group). Similar absolute improvements in compared with placebo were observed in subgroups with screening levels lower than 70 and levels of 70 or higher. Increases in body-mass index and reduction in number of pulmonary exacerbion events were observed in both lumacaftor/ivacaftor dose groups compared with placebo across all lung function subgroups. Trement was generally well tolered, although the incidence of some respirory adverse events was higher with lumacaftor/ivacaftor than with placebo in all subgroups. In pients with levels lower than 40, these adverse events included cough, dyspnoea, and abnormal respirion. Lancet Respir Med 2016; 4: Published Online June 10, S (16) See Comment page 594 Members listed in the appendix Queen s University Belfast, Belfast, UK (Prof J S Elborn MD); Setle Children s Hospital, University of Washington School of Medicine, Cystic Fibrosis Clinic, Setle, WA, USA (Prof B W Ramsey MD); Johns Hopkins Medical Institutions, Sheikh Zayed Tower, Baltimore, MD, USA (Prof M P Boyle MD); Case Western Reserve University School of Medicine, Rainbow Babies and Children s Hospital, Cleveland, OH, USA (M W Konstan MD); Vertex Pharmaceuticals Incorpored, Boston, MA, USA (X Huang PhD, G Marigowda MD, D Waltz MD); and School of Medicine, University of Queensland, South Brisbane, QLD, Australia (Prof C E Wainwright MD) Correspondence to: Prof J Stuart Elborn, Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen s University, Belfast BT9 7BL, UK s.elborn@qub.ac.uk See Online for appendix Interpretion These analyses confirm th lumacaftor/ivacaftor combinion therapy benefits pients with cystic fibrosis homozygous for Phe508del CFTR who have varying degrees of lung function impairment. Funding Vertex Pharmaceuticals. Introduction The most common cystic fibrosis-causing mution, Phe508del in the cystic fibrosis transmembrane conductance regulor (CFTR) gene, leads to various defects, including reduced folding and trafficking of the CFTR protein to the epithelial cell surface and defective channel ging, among others. 1 4 Therefore, restoring the chloride transport activity of the Phe508del CFTR channel is complex. Lumacaftor is a CFTR corrector, which selectively increases the processing and trafficking of Phe508del CFTR to the cell surface and enhances CFTR-medied chloride transport in vitro. 5 Ivacaftor is a CFTR potentior, which facilites chloride transport by increasing the channel-open probability of CFTR on the Vol 4 August

2 Research in context Evidence before this study We searched PubMed on April 12, 2016, for the terms ivacaftor or VX-770, lumacaftor or VX-809, and clinical trial, with no restrictions on publicion de or language, and retrieved three relevant clinical studies. In phase 2 studies, combinion lumacaftor/ivacaftor therapy, but not monotherapy, improved lung function and had an acceptable side-effect profile in pients with cystic fibrosis homozygous for the Phe508del CFTR mution. The phase 3 TRAFFIC and TRANSPORT studies showed a clinically ingful benefit of lumacaftor/ivacaftor combinion therapy in this populion. To be eligible for these studies pients had to have a screening percent predicted FEV 1 ( ) of Therefore, few da are available on which to base trement decisions in pients whose is lower than 40. Added value of this study We assessed the response to lumacaftor/ivacaftor therapy in the phase 3 TRAFFIC and TRANSPORT studies in pients with cystic fibrosis homozygous for the Phe508del CFTR mution strified by specific cegories of lung function, including a subgroup of pients with severe lung dysfunction whose declined to less than 40 between screening and. This provided an opportunity to assess the response in this group of pients th is often not studied. Results of this prespecified subgroup analysis provide evidence th lumacaftor/ivacaftor therapy improved levels in pients across a spectrum of pretrement lung function. The incidence of some respirory adverse events was higher in pients whose was lower than 40 than in those with a of 40 or higher. Across lung function subgroups, some respirory adverse events occurred more frequently in pients who received lumacaftor/ivacaftor therapy than placebo. These respirory adverse events were associed with the initiion of trement, irrespective of lung function subgroup, and usually resolved with continued trement. Discontinuions because of adverse events were low and similarly frequent across subgroups. Implicions of all the available evidence These da show th lumacaftor/ivacaftor combinion therapy benefits pients with cystic fibrosis homozygous for the Phe508del CFTR mution with varying degrees of lung function impairment, including those with modere to severe dysfunction. Prospective assessment is warranted in pients with values lower than 40, particularly in those with values lower than 30, in whom the safety and efficacy of lumacaftor/ivacaftor combinion therapy are currently being assessed. cell surface. 6 Monotherapy with either lumacaftor or ivacaftor was not shown to be clinically beneficial in pients with cystic fibrosis homozygous for the Phe508del CFTR mution. 7,8 By contrast, clinically ingful benefits were observed with combinion therapy with lumacaftor/ivacaftor, in pients with cystic fibrosis homozygous for the Phe508del CFTR mution in a phase 2 and in two phase 3, randomised, doubleblind, placebo-controlled trials, TRAFFIC and TRANSPORT. 9,10 Significant improvements in lung function were observed with lumacaftor 600 mg once daily/ivacaftor 250 mg every 12 h and lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h in the TRAFFIC and TRANSPORT studies; the absolute change in percent predicted FEV 1 ( ) week 24 versus placebo ranged from 2 8 to 3 3 percentage points in the pooled analysis (p<0 001). 10 Improvements were also observed in nutritional stus and re of pulmonary exacerbions. These da supported the approval of lumacaftor/ivacaftor combinion therapy (Orkambi, Vertex Pharmaceuticals, MA, USA) in pients aged 12 years and older with cystic fibrosis homozygous for the Phe508del CFTR mution in the USA, the European Union, and Canada. Pients with cystic fibrosis whose is in the severe range have a greer burden of disease associed with a higher re of pulmonary exacerbions and worse nutritional stus. 11,12 The safety and efficacy of new trements in pients with severe lung dysfunction might not be the same as in pients with milder dysfunction. The TRAFFIC and TRANSPORT studies enrolled pients with values of 40 to 90 screening, reflecting a range of lung function impairment from mild ( 70 to 90) to modere ( 40 to 69). Some pients had a value th decreased to below 40 between screening and, providing an opportunity to assess trement response in this clinically important subgroup. 10 Prospective assessment of the safety and efficacy of lumacaftor/ ivacaftor in pients with severe lung dysfunction is ongoing. We did a prespecified pooled analysis of da from the TRAFFIC and TRANSPORT studies to assess the efficacy and safety of lumacaftor/ivacaftor combinion therapy in pients with cystic fibrosis homozygous for the Phe508del CFTR mution, defined by specific cegories of lung function, including those with severe lung dysfunction ( <40 ). Methods Study design and pients Both trials (TRAFFIC and TRANSPORT) included in this pooled analysis were multinional, randomised, double-blind, placebo-controlled, parallel-group, 24-week phase 3 studies done between April, 2013, and April, The design of these nearly identical studies has been Vol 4 August 2016

3 described previously 10 and is briefly reviewed in the appendix. The studies, which were done 187 sites in North America, Australia, and the European Union, included pients aged 12 years or older with a confirmed diagnosis of cystic fibrosis, homozygous for the Phe508del CFTR mution, and with a of the time of screening. Some pients had levels th decreased to below 40 between the screening and visits ( 4 weeks). In this analysis, da from the two studies were pooled by dosing regimens. Both trials were done in accordance with the principles of the Declarion of Helsinki and in compliance with Good Clinical Practice guidelines and all applicable local and nional regulions. The study protocol for both trials was approved by ethics committees and institutional review boards approprie for each country and institution, and all pients provided written informed consent. Randomision and masking Pients were randomly assigned (1:1:1) using an interactive web response system to one of three study groups: 600 mg of lumacaftor once daily in combinion with 250 mg of ivacaftor every 12 h (lumacaftor [600 mg/day] ivacaftor), 400 mg of lumacaftor every 12 h in combinion with 250 mg of ivacaftor every 12 h (lumacaftor [400 mg/12 h] ivacaftor), or lumacaftormched placebo every 12 h in combinion with ivacaftor-mched placebo every 12 h. Randomision was strified according to age (<18 years vs 18 years), sex, and pulmonary function ( ) screening. Both participants and assessors were blinded to trement assignment in both trials. Outcomes For the pooled TRAFFIC and TRANSPORT study da, we did preplanned subgroup analyses of levels lower than 40 versus 40 or higher and levels lower than 70 versus 70 or higher screening for the primary endpoint and key secondary endpoints in a manner similar to th reported previously for the entire study cohort. 10 The primary endpoint was the absolute change from in week 24, calculed by averaging the absolute change week 16 and the absolute change week 24 because this was anticiped to provide a better estime of the trement difference compared with placebo the end of the trement period given the inherent variability in. The main secondary endpoints were the relive change from in week 24 (calculed by averaging the relive change weeks 16 and 24); the percentage of pients with least a 5% relive increase from in (response derived using average relive change weeks 16 and 24); the absolute change from in body-mass index week 24; the absolute change from in the Cystic Fibrosis Questionnaire-Revised (CFQ-R) respirory domain score week 24; and the number of pulmonary exacerbions through to week 24 (expressed as a re over 48 weeks). Additionally, post-hoc subgroup analyses were done for the absolute change from in each study visit, the percentage of pients with least a 10% relive increase from in (response derived using average relive change weeks 16 and 24), the number of pulmonary exacerbions requiring intravenous antibiotics, the number of pulmonary exacerbions requiring hospital admission, and the number needed to tre to prevent a pulmonary exacerbion during the 24-week study period. The number needed to tre indices the number of pients who would need to be treed to prevent one additional event. Safety and tolerability were assessed by reports of adverse events and by clinical laborory variables. Stistical analysis The primary endpoint was measured in all randomised pients. The efficacy analysis populion included all pients who were randomised and received least one dose of study drug (full analysis set). Pients were analysed according to the study group to which they were randomly assigned. Pooled da were analysed for each subgroup separely, defined according to (<40 and 40) and screening (<70 and 70); these subgroup analyses were not powered stistically for efficacy comparisons between trement groups. We considered p values lower than 0 05 as stistically significant and did not adjust for multiple comparisons. The least-squares s for the subgroup analysis of the absolute and relive changes from Placebo overall (n=371) Lumacaftor/ivacaftor overall <40 (n=53) 40 (n=678) <70 (n=527) 70 (n=204) Female 181 (49%) 31 (58%) 331 (49%) 269 (51%) 93 (46%) Median age (years) 23 0 (12 64) 27 0 (13 44) 23 0 (12 57) 26 0 (12 57) 18 5 (12 53) Mean 60 4 ( ) 37 2 ( ) 62 5 ( ) 54 0 ( ) 77 9 ( ) Body-mass index (mg/kg²) 21 0 (2 9) 20 9 (3 4) 21 3 (3 0) 21 2 (2 9) 21 4 (3 3) Chronic cystic fibrosis therapy use Bronchodilors (any) 342 (92%) 50 (94%) 631 (93%) 496 (94%) 185 (91%) Dornase alfa 281 (76%) 41 (77%) 517 (76%) 407 (77%) 151 (74%) Inhaled antibiotic 258 (70%) 33 (62%) 421 (62%) 351 (67%) 103 (50%) Inhaled hypertonic 220 (59%) 34 (64%) 386 (57%) 294 (56%) 126 (62%) saline Inhaled corticosteroids 220 (59%) 35 (66%) 386 (57%) 311 (59%) 110 (54%) Da are n (%), median (range), (range), or (SD). = percent predicted FEV pients (28 in the placebo group and 53 in the lumacaftor/ivacaftor group) had a th decreased to lower than 40 between screening and. Da shown are for pients with <70 and 70. Table 1: Baseline characteristics Vol 4 August

4 Absolute change in Within group least-squares (percentage points) placebo, 95% CI (percentage points) Placebo <40 (n=28) 0 4 (1 3) 40 (n=338) 0 4 (0 4) Lumacaftor 600 mg daily/ivacaftor 250 mg every 12 h <40 (n=24) 3 7 (0 5 to 6 9) 40 (n=342) Lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h <40 (n=29) 3 3 (2 3 to 4 4) 3 3 (0 2 to 6 4) 40 (n=336) 2 8 (1 7 to 3 8) p value < < Relive change in Within group least-squares 1 5% (3 4) 0 2% (0 7) placebo, 95% CI 9 9% (1 2 to 18 5) 5 3% (3 5 to 7 1) 9 1% (0 7 to 17 4) 4 5% (2 7 to 6 3) p value < < Relive increase of 5% from in OR vs placebo 2 4 (0 8 to 7 2) 3 1 (2 2 to 4 3) 1 7 (0 6 to 5 2) 2 3 (1 6 to 3 2) p value < < Body-mass index Within group least-squares 0 1 (0 2) 0 1 (0 1) placebo, 95% CI (kg/m²) 0 6 (0 1 to 1 2) 0 3 (0 1 to 0 4) 0 3 ( 0 2 to 0 8) 0 2 (0 1 to 0 4) p value CFQ-R respirory domain Within group least-squares 5 8 (3 2) 0 9 (0 9) placebo, 95% CI (points) 3 3 ( 5 2 to 11 7) 3 3 (1 0 to 5 7) 4 2 ( 12 0 to 3 7) 2 9 (0 5 to 5 3) p value Da are least-squares (SE) or OR (95% CI), unless otherwise specified. = percent predicted FEV 1. SE=standard error. OR=odds rio. CFQ-R=Cystic Fibrosis Questionnaire-Revised. was measured ; 81 pients had levels th decreased to lower than 40 between screening and. Assessed by averaging the values from weeks 16 and 24, as prespecified in the stistical analysis plan. Average relive increase from weeks 16 and 24. Table 2: Efficacy results after trement with lumacaftor/ivacaftor for 24 weeks in pients with lower than 40 versus 40 or higher in were calculed with a mixed-effects model for repeed measures th included study, sex, age (<18 years vs 18 years), trement, visit, and trement-by-visit interaction. The odds rio versus placebo for the percentage of pients with least a 5% and least a 10% relive increase from in for each subgroup was estimed with the Cochran-Mantel-Haenszel test, strified by study, age (<18 years vs 18 years), and sex. The leastsquares s for the subgroup analysis of absolute change in body-mass index and CFQ-R respirory domain were calculed with a mixed-effects model for repeed measures th included study, sex, age, trement, visit, and trement-by-visit interaction, plus the corresponding as a covarie. The re rio of pulmonary exacerbion events for each subgroup (ie, event re per year for the trement group vs th for the placebo group) was calculed with a negive binomial regression model th included study, trement, sex, and age, with log (time in study in years) as an offset; 48 weeks was considered equivalent to 1 year for the analysis. The safety analysis included all pients who received any amount of study drug and was based on actual trement received. Pients who received medicion from more than one trement group during the studies were considered to be in the lower dose of the active trement group. Stistical analyses were done with stistical software SAS version 9.2 or higher. Both trials are registered with ClinicalTrials.gov (TRAFFIC: NCT ; TRANSPORT: NCT ). Role of the funding source The funder participed in the design of the protocol, did the stistical analysis, and was involved in da interpretion. Medical writing and editorial support and coordinion were provided by the funder. All authors had full access to the study da. The corresponding author contributed to da interpretion and manuscript conception, writing and revision, and made the final decision to submit for publicion. Results 1449 pients were screened for the two trials. Of these, 1122 pients were randomised in the TRAFFIC and TRANSPORT studies, done between April, 2013, and April, Of these, 1108 received least one dose of study trement (three pients in the placebo group, four in the lumacaftor [600 mg/day] ivacaftor group, and seven in the lumacaftor [400 mg/12 h] ivacaftor group did not receive study drug). 342 pients (31%) had a of 70 or higher screening, and 730 (66%) had a of lower than 70 screening pients (92%) had a of more than 40 ; 81 pients (7%) had a level th decreased to less than 40 between the screening and visits (range ). In the pooled da, trement groups were well balanced across demographic and characteristics (table 1), as reported previously. 10 A high percentage of pients in each subgroup reported maintenance use of bronchodilors and multiple other cystic fibrosis trements. Most pients in each subgroup completed 24 weeks of study trement, including 78 (96%) of the 81 pients with severe lung dysfunction ( <40). Significant improvements in the primary efficacy endpoint, absolute change from in week 24, were observed with both doses of lumacaftor/ ivacaftor in the subgroup with levels lower than 40 (least-squares difference vs placebo in the lumacaftor [600 mg/day] ivacaftor group was 3 7 percentage points [95% CI ; p=0 024] and in Vol 4 August 2016

5 the lumacaftor [400 mg/12 h] ivacaftor group was 3 3 percentage points [ ; p=0 036]) and in the subgroup with levels of 40 or higher (3 3 percentage points [ ; ] and 2 8 percentage points [ ; ]; table 2). Generally similar results favouring lumacaftor/ivacaftor over placebo were observed in subgroups with levels lower than 70 and 70 or higher screening, although stistical significance was not reached in the subgroup with levels of 70 or higher receiving lumacaftor (400 mg/12 h) ivacaftor (table 3). The absolute change versus placebo across all lung function subgroups ranged from 1 9 to 3 7 percentage points, which was consistent with differences observed in the overall populion pooled from the two studies by dosing regimen ( percentage points). 10 The absolute change from in each study visit throughout 24 weeks of trement in subgroups defined by is shown in figure 1. Improvements in were observed as early as day 15 and were sustained through week 24 with both lumacaftor/ivacaftor doses in these subgroups. The differences between lumacaftor/ivacaftor and placebo with respect to relive change from week 24 in were consistent with results for the absolute change in. Relive improvements in with lumacaftor (600 mg/day) ivacaftor and lumacaftor (400 mg/12 h) ivacaftor versus placebo were observed in the subgroups with levels lower than 40 and 40 or higher (table 2). Relive improvements in with both lumacaftor/ivacaftor doses versus placebo were also observed in the subgroups with screening levels lower than 70 and 70 or higher; however, significance was not reached in the group with levels of 70 or higher receiving lumacaftor (400 mg/12 h) ivacaftor (table 3). The proportion of pients with 5% or higher and 10% or higher average relive increases from weeks 16 and 24 in was significantly higher with both lumacaftor/ivacaftor doses than with placebo in subgroups with levels of 40 or more (p 0 002) and levels of lower than 70 screening (p ; figure 2). Similar trends favouring lumacaftor/ivacaftor doses were observed in the other subgroups, but stistical significance was not reached in most comparisons in the smaller subgroup with levels lower than 40; significance was achieved for most comparisons in the subgroup with screening levels of 70 or higher (figure 2). For other clinical variables, we observed generally consistent improvements across subgroups. The absolute change in body-mass index was stistically significant in most subgroups (tables 2, 3). Improvements in the CFQ-R respirory domain score favouring lumacaftor/ivacaftor over placebo were observed in some of the larger subgroups, including those with levels of 40 or higher both lumacaftor/ivacaftor doses (tables 2, 3), although Placebo <70 (n=244) 70 (n=109) Lumacaftor 600 mg daily/ivacaftor 250 mg every 12 h <70 (n=241) 70 (n=119) variability on this measure was high, particularly in the subgroups with small pient numbers. Trement with lumacaftor/ivacaftor significantly reduced the number of pulmonary exacerbions compared with placebo in most subgroups (tables 4, 5). Additionally, trends toward fewer pulmonary exacerbion events requiring intravenous antibiotic therapy and hospital admissions were observed in both lumacaftor/ivacaftor dose groups versus placebo across all lung function subgroups (tables 4, 5). To further characterise these findings, an analysis of the number needed to tre was done based on those pients who had a pulmonary exacerbion. The number needed to tre to prevent a pulmonary exacerbion in pients treed with lumacaftor (600 mg/day) ivacaftor and Lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h <70 (n=245) Absolute change in Within group leastsquares (percentage points) 0 5 (0 4) 0 1 (0 8) placebo, 95% CI, (percentage points) 3 3 (2 1 to 4 4) 3 3 (1 3 to 5 4) 3 3 (2 1 to 4 4) 70 (n=114) 1 9 ( 0 2 to 4 0) p value < < Relive change in Within group leastsquares 0 3% (0 9) 0 7% (1 1) placebo, 95% CI 6 0% (3 7 to 8 2) 4 4% (1 5 to 7 4) 5 9% (3 6 to 8 2) 2 5% ( 0 5 to 5 5) p value < < Relive increase of 5% from in OR vs placebo (95% CI) 2 5 (1 7 to 3 7) 3 8 (2 1 to 6 8) 2 4 (1 6 to 3 5) 1 9 (1 0 to 3 4) p value < < < Body-mass index Within group leastsquares 0 1 (0 1) 0 1 (0 1) placebo (kg/m²) 0 2 (0 0 to 0 4) 0 4 (0 2 to 0 7) 0 2 (0 0 to 0 3) 0 3 (0 1 to 0 6) p value CFQ-R respirory domain Within group leastsquares 1 5 (1 1) 1 7 (1 4) placebo, 95% CI (points) 4 1 (1 3 to 6 9) 1 9 ( 1 9 to 5 7) 1 9 ( 0 9 to 4 7) 3 6 ( 0 3 to 7 4) p value Da are least-squares (SE) or OR (95% CI), unless otherwise specified. = percent predicted FEV 1. SE=standard error. OR=odds rio. CFQ-R=Cystic Fibrosis Questionnaire-Revised. are measured screening; 81 pients had levels th decreased to lower than 40 between screening and. Assessed by averaging the values from weeks 16 and 24, as prespecified in the stistical analysis plan. Average relive increase from weeks 16 and 24. Table 3: Efficacy results after trement with lumacaftor/ivacaftor for 24 weeks in pients with lower than 70 versus 70 or higher screening Vol 4 August

6 Absolute change in (percentage points), least-squares A Lumacaftor (600 mg/day) ivacaftor Lumacaftor (400 mg/12 h) ivacaftor Placebo B C 10 D Absolute change in (percentage points), least-squares Baseline Day 15 Week 4 Week 8 Week 16 Week 24 Timepoint Average of weeks 16 and 24 Baseline Day Week 15 4 Week 8 Week 16 Week 24 Timepoint Average of weeks 16 and 24 Figure 1: Absolute change from in each study visit for pients with levels lower than 40 (A) or 40 or higher (B) and lower than 70 (C) or 70 or higher (D) screening Error bars show 95% CI. =percent predicted FEV 1. A p=0 331 p=0 113 Placebo Lumacaftor (600 mg/day) ivacaftor Lumacaftor (400 mg/12 h) ivacaftor p=0 084 p=0 037 B p=0 045 Proportion of pients (%) p=0 002 Proportion of pients (%) p= p= p=0 251 p= < <40 40 <70 70 < % relive change 10% relive change 5% relive change 10% relive change thresholds by subgroup (%) thresholds by subgroup (%) Figure 2: Percentage of pients with 5% or higher and 10% or higher average relive increases from in weeks 16 and 24 in pients with levels lower than 40 or 40 or higher (A) and levels lower than 70 or 70 or higher screening (B) =percent predicted FEV Vol 4 August 2016

7 lumacaftor (400 mg/12 h) ivacaftor versus placebo was 5 3 and 8 0, respectively, in the subgroup with levels lower than 40 and 10 5 and 6 8, respectively, in the subgroup with levels of 40 or higher. The number needed to tre to prevent a pulmonary exacerbion in both lumacaftor/ivacaftor dose groups versus placebo was 12 3 and 8 0, respectively, in the subgroup with screening levels lower than 70 and 5 9 and 5 4, respectively, in the subgroup with screening levels of 70 or higher. The overall incidence of adverse events in both lumacaftor/ivacaftor groups and in the placebo group was similar in pients with levels lower than 40 and 40 or higher and those with levels lower than 70 and 70 or higher screening (table 6). Because the incidence of adverse events was similar between the two lumacaftor/ivacaftor dose groups, the safety da of the two dosing regimens were pooled. The most commonly reported adverse events across all trement groups were infective pulmonary exacerbions of cystic fibrosis and cough. The incidence of some respirory adverse events was greer in the pooled lumacaftor/ivacaftor group than in the placebo group in all subgroups; in pients with levels lower than 40, these adverse events with higher incidence in the pooled lumacaftor/ ivacaftor group than in placebo included cough (21 [40%] of 53 vs 7 [25%] of 28), dyspnoea (14 [26%] of 53 vs 4 [14%] of 28), and abnormal respirion (the preferred term for chest tightness; 4 [8%] of 53 vs 1 [4%] of 28). The incidence of dyspnoea and abnormal respirion was also greer in the pooled lumacaftor/ivacaftor group than in the placebo group in those with levels of 40 or higher, and in those with screening levels lower than 70 and 70 or higher (table 6). Irrespective of lung function subgroup, respirory adverse events were associed with the initiion of trement and usually resolved with continued trement. The median time (minimum maximum) to onset of the first adverse event of special interest of respirory symptoms was 2 days (1 170) for the pooled lumacaftor/ivacaftor groups (n=738) and 43 days (1 172) for the placebo group (n=370). Generally similar results were observed across subgroups, with a shorter time to onset of the first adverse event of special interest of respirory symptoms in lumacaftor/ ivacaftor-treed pients in all subgroups. The median time to onset of first adverse event of respirory symptoms in the subgroup with levels lower than 40 was 1 day (range 1 155) for the pooled lumacaftor/ivacaftor groups (n=18) and 7 days (2 43) in the placebo group (n=5); in the subgroup with levels of 40 or higher, the median time was 2 days (1 170) for the pooled lumacaftor/ivacaftor groups (n=150) and 43 days (1 172) in the placebo group (n=46). With respect to values, the incidence of dyspnoea was approximely two times higher in pients Lumacaftor 600 mg daily/ivacaftor 250 mg every 12 h <40 (n=24) 40 (n=342) with levels lower than 40 versus levels of 40 or higher in both the placebo group and the lumacaftor/ ivacaftor group, which was consistent with wh might be expected for a populion of pients with more severe lung dysfunction (table 6). The incidence of dyspnoea was also increased in pients with screening levels lower than 70 versus 70 or higher in both the placebo group and active trement group (table 6). The incidence of cough was greer in pients with levels lower than 40 versus 40 or higher in the lumacaftor/ ivacaftor group but lower in the placebo group (table 6). The incidence of cough in pients with screening levels lower than 70 versus 70 or higher was generally similar in both the placebo group and the lumacaftor/ ivacaftor group (table 6). The proportion of pients who discontinued trement because of adverse events was small across all subgroups; such discontinuions occurred in 1 (4%) pient who received placebo and zero Lumacaftor 400 mg every 12 h/ ivacaftor 250 mg every 12 h <40 (n=29) 40 (n=336) Pulmonary 0 47 ( ) 0 73 ( ) 0 59 ( ) 0 61 ( ) exacerbion events p value < Events requiring 0 41 ( ) 0 57 ( ) 0 56 ( ) 0 42 ( ) intravenous antibiotic therapy p value < Events requiring 0 43 ( ) 0 63 ( ) 0 67 ( ) 0 36 ( ) hospital admission p value < Da are re rio (95% CI), unless otherwise specified. = percent predicted FEV 1. calculed ; 81 pients had th decreased to lower than 40 between screening and Table 4: Pulmonary exacerbion events through to week 24 by subgroup and trement group Lumacaftor 600 mg daily/ivacaftor 250 mg every 12 h <70 (n=241) 70 (n=119) Lumacaftor 400 mg every 12 h/ ivacaftor 250 mg every 12 h <70 (n=245) 70 (n=114) Pulmonary 0 74 ( ) 0 55 ( ) 0 65 ( ) 0 51 ( ) exacerbion events p value Events requiring 0 53 ( ) 0 53 ( ) 0 49 ( ) 0 22 ( ) intravenous antibiotic therapy p value < < Events requiring 0 59 ( ) 0 53 ( ) 0 48 ( ) 0 09 ( ) hospital admission p value Da are re rio (95% CI), unless otherwise specified. = percent predicted FEV 1. calculed screening. Table 5: Pulmonary exacerbion events through to week 24 by screening subgroup and trement group Vol 4 August

8 Placebo Lumacaftor/ivacaftor Placebo Lumacaftor/ivacaftor <40 (n=28) 40 (n=337) <40 (n=53) 40 (n=679) screening <70 (n=243) 70 (n=109) <70 (n=487) 70 (n=233) Pients who had any adverse event 28 (100%) 322 (96%) 52 (98%) 649 (96%) 235 (97%) 102 (94%) 466 (96%) 224 (96%) Adverse events reported in 10% of pients in any subgroup of placebo or total lumacaftor/ivacaftor Infective pulmonary exacerbions of 20 (71%) 162 (48%) 27 (51%) 248 (37%) 125 (51%) 53 (49%) 211 (43%) 59 (25%) cystic fibrosis Cough 7 (25%) 140 (42%) 21 (40%) 203 (30%) 94 (39%) 47 (43%) 153 (31%) 68 (29%) Dyspnoea 4 (14%) 25 (7%) 14 (26%) 88 (13%) 26 (11%) 3 (3%) 83 (17%) 17 (7%) Increased sputum 8 (29%) 62 (18%) 13 (25%) 94 (14%) 49 (20%) 18 (17%) 80 (16%) 25 (11%) Headache 5 (18%) 52 (15%) 10 (19%) 103 (15%) 42 (17%) 14 (13%) 74 (15%) 36 (15%) Pyrexia 5 (18%) 29 (9%) 8 (15%) 59 (9%) 28 (12%) 6 (6%) 51 (10%) 15 (6%) Diarrhoea 2 (7%) 29 (9%) 7 (13%) 73 (11%) 19 (8%) 10 (9%) 62 (13%) 16 (7%) Nausea 3 (11%) 25 (7%) 7 (13%) 67 (10%) 18 (7%) 9 (8%) 56 (11%) 17 (7%) Figue 2 (7%) 27 (8%) 6 (11%) 57 (8%) 21 (9%) 7 (6%) 48 (10%) 15 (6%) Haemoptysis 7 (25%) 43 (13%) 6 (11%) 95 (14%) 42 (17%) 8 (7%) 81 (17%) 18 (8%) Nasopharyngitis 2 (7%) 37 (11%) 6 (11%) 65 (10%) 30 (12%) 8 (7%) 49 (10%) 20 (9%) Oropharyngeal pain 1 (4%) 29 (9%) 6 (11%) 61 (9%) 17 (7%) 11 (10%) 43 (9%) 24 (10%) Upper respirory tract infection 0 19 (6%) 6 (11%) 53 (8%) 12 (5%) 5 (5%) 39 (8%) 18 (8%) Nasal congestion 1 (4%) 43 (13%) 5 (9%) 52 (8%) 22 (9%) 21 (19%) 34 (7%) 23 (10%) Abnormal respirion 1 (4%) 21 (6%) 4 (8%) 68 (10%) 19 (8%) 2 (2%) 49 (10%) 22 (9%) Blood creinine phosphokinase 1 (4%) 19 (6%) 2 (4%) 39 (6%) 7 (3%) 12 (11%) 24 (5%) 16 (7%) increased Viral upper respirory tract infection 4 (14%) 20 (6%) 2 (4%) 48 (7%) 15 (6%) 8 (7%) 34 (7%) 16 (7%) = percent predicted FEV 1. Pooled da for the lumacaftor 600 mg daily/ivacaftor 250 mg every 12 h and lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h groups. Table 6: Summary of trement-emergent adverse events pients who received lumacaftor/ivacaftor in the subgroup with levels lower than 40, and in 5 (2%) pients who received placebo and 31 (5%) pients who received lumacaftor/ivacaftor in the subgroup with levels of 40 or higher. Discussion This pooled analysis of da from the TRAFFIC and TRANSPORT studies shows th the efficacy and safety of lumacaftor/ivacaftor in pients with cystic fibrosis homozygous for the Phe508del CFTR mution was similar across lung function subgroups, including levels lower than 40 and 40 or higher and levels lower than 70 and 70 or higher screening. The da in the subgroup with levels lower than 40 were notable given the severity of lung function impairment in these pients ( range of ). In this subgroup, the absolute improvement in lung function, as measured by, from week 24 with both lumacaftor/ivacaftor doses compared with placebo, ranged from 3 3 to 3 7 percentage points, which was similar to the improvement in lung function observed in those with levels of 40 or higher ( percentage points) and in the overall study populion. 10 Also notable were outcomes in pients whose levels were 70 or higher screening; lung function improvements in this subgroup were also generally consistent with the overall study populion. 10 Clinical improvements in body-mass index were also seen with both lumacaftor/ivacaftor doses compared with placebo; these were generally similar in magnitude across lung function subgroups. Furthermore, clinically ingful reductions in pulmonary exacerbion events were observed across lung function subgroups, including those with levels lower than 40 and 70 or higher screening. Similarly, reductions in those events requiring the use of intravenous antibiotics and hospital admission were observed across subgroups; most of these comparisons reached stistical significance. Although the small sample size in some subgroups limits the strength of our findings, the differences in the main outcome measure were stistically significant; the finding th the effect sizes are consistent with a ingful clinical benefit in these subgroups is encouraging. The number needed to tre calculion showed variability across the two doses, but those with a level lower than 40 had a similar number needed to tre to those with a level of 40 or higher. With the use of the respirory domain of the CFQ-R, a cystic fibrosis-specific pient-reported outcome Vol 4 August 2016

9 instrument, 13 significant improvements were noted in some of the subgroups with larger pient numbers, with consistent trends in other subgroups, with the exception of the subgroup with levels of lower than 40 treed with the lumacaftor (400 mg/12 h) /ivacaftor dose. A possible explanion for this finding could be reled to adverse events; however, variability in this measure was high, particularly in the smaller subgroups, which limited interpretion of these findings. The side-effect profile of lumacaftor/ivacaftor therapy was acceptable in each lung function subgroup. The res of discontinuion because of adverse events were low across lung function subgroups. The incidence of respirory adverse events (such as dyspnoea) was higher in subgroups with more impaired lung function (eg, levels lower than 40 vs 40 or higher) in both the placebo and lumacaftor/ivacaftor groups. The increased incidence of some respirory adverse events in those with levels lower than 40 versus 40 or higher is consistent with the nure of cystic fibrosis in a populion of pients with more severe lung dysfunction. The incidence of some respirory adverse events was also higher in the active trement groups versus placebo groups, notably in the subgroup with levels lower than 40 (eg, dyspnoea and abnormal respirion, or chest tightness). When respirory adverse events were present, they were generally associed with the initiion of trement, irrespective of lung function impairment, and usually resolved with continued trement. These subgroup analyses were not powered stistically for efficacy comparisons between trement groups. This is particularly important for subgroups with small numbers of pients, such as those with levels lower than 40. Nevertheless, the outcomes in pients with severe lung dysfunction were consistent with improvements observed in pients with levels of 40 or higher, suggesting a benefit of lumacaftor/ivacaftor combinion therapy across a range of differing values. The generalisability of these findings to pients with severe lung dysfunction should be approached cautiously because these trials were not designed to recruit pients with levels below 40. Perhaps, these pients might not fully reflect the profile of pients specifically recruited on the basis of having levels lower than 40. Prospective assessment is needed to confirm the benefit of lumacaftor/ivacaftor therapy in this clinically important subgroup. Further, we consider the possibility th selection bias might have affected the pients enrolled in the study, particularly in the subgroup with levels lower than 40, as physicians might have been motived to optimise the health of their pients whose lung function was near the eligibility cutoff. Although this would not be expected to affect efficacy outcomes because of the randomised nure of the trial, a potential effect on safety outcomes cannot be discounted. Finally, the subgroup of pients with severe lung dysfunction included in this analysis had values ranging between 31 1 and 39 9; therefore, special tention might be needed in initiing pients with below 30 until further results are available. An open-label phase 3b trial to assess the safety and efficacy of lumacaftor/ivacaftor combinion therapy in pients with severe lung dysfunction is currently ongoing (ClinicalTrials.gov number, NCT ). Overall, the results of these subgroup analyses of the phase 3 TRAFFIC and TRANSPORT studies showed generally consistent improvements across lung function subgroups, including those with lower than 40 and 70 or higher, suggesting th lumacaftor/ivacaftor combinion therapy was generally well tolered and benefits pients with cystic fibrosis homozygous for the Phe508del CFTR mution across a spectrum of lung function impairment. Contributors JSE contributed to the study design, collection, analysis, and interpretion of da, and drafting of the paper. BWR contributed to the study design, interpretion of da, and drafting of the paper. MPB contributed to the collection, analysis, and interpretion of da, and drafting of the paper. MWK contributed to the interpretion of da and drafting of the paper. XH contributed to the study design, analysis and interpretion of da, and drafting of the paper. GM contributed to the study design, collection, analysis, and interpretion of da. DW contributed to study design, analysis, and interpretion of da, and drafting of the paper. CEW contributed to the interpretion of da and critical review of the paper. Declarion of interests JSE reports speaker fees from Vertex Pharmaceuticals, grants from Novartis and ProQR, and consultant fees from ProQR during the conduct of the study. BWR reports contract support from Aridis, Celtaxsys, Flley Discover Lab LLV, KaloBios, Laurent Therapeutics, Nilvalis Therapeutics, Synedgen, and Vertex Pharmaceuticals outside of the submitted work. MPB reports grants from Vertex Pharmaceuticals during the conduct of the study. MWK reports grants, consultant fees, and travel support from Vertex Pharmaceuticals during the conduct of the study; grants and travel support from the Cystic Fibrosis Foundion; consultant fees from Anthera, Chiesi, Digestive Carec, and Laurent; grants, consultant fees, and travel support from Genentech, Insmed, Novartis, PTC Therapeutics, and Vertex Pharmaceutical Incorpored; consultant fees and travel support from AbbVie, Celtaxsys, and Gilead; grants and consultant fees from Savara and KaloBios, outside of the submitted work. XH, GM, and DW are employees of Vertex Pharmaceuticals and might own stock or stock options in th company. CEW reports receiving grant income on a per pient basis for conducting studies, consultant fees, and travel support from Vertex Pharmaceuticals during conduct of the study; grant income on a per pient basis from Vertex Pharmaceuticals, Boehringer Ingelheim, and Ablynx NV outside of the submitted work; and a research grant from Novo Nordisk; and honoraria and travel support from Novartis outside of the submitted work. Acknowledgments Editorial coordinion and support were provided by Dhrupad Pel, an employee of Vertex Pharmaceuticals who might own stock or stock options in th company. Additional stistical analysis was provided by Minoo Niknian, an employee of Vertex Pharmaceuticals Incorpored who might own stock or stock options in th company. Medical writing and editorial support were provided by Michelle Yochum and Dena McWain. Michelle Yochum and Dena McWain are employees of Infusion Communicions, which received funding from Vertex Pharmaceuticals. Vol 4 August

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